Fluorescent dye as a functional dye has been widely used in various fields, and attracts much attention on the research especially in life science, clinical diagnosis, fluorescent immune analysis and detection, and so on. In cell biology, fluorescence spectrum has been used in tracking of position and migration of cellular compositions. Moreover, recognition and assortment of cells also relies on the flow cytometry which is based on fluorescent technology. Therefore, the development of fluorescent dye with excellent photophysical properties is crucial to fluorescent analysis technology.
By comparison of the other fluorescent dye, cyanine fluorescent dye as a member of fluorescent dyes has prominent advantages in the application fields of ion probe, bio-labeling (DNA and protein labeling), and live cell and vivo tissue imaging, such as large molar extinction coefficient and increased maximum absorption and emission wavelengths upon elongation of conjugated chain. For example, maximum absorption and emission wavelengths of pentamethine and heptamethine cyanine dyes are above 600 nm, particularly for heptamethine cyanine dye, absorption and emission wavelengths are above 750 nm which are close to near-Infrared area, which can effectively avoid autofluorescence from bio-tissues.
Generally, Stokes shift of cyanine dye is about 20 nm, which is disadvantageous for the bioapplication of the dye. This is because small Stokes shift would cause some problems as follows:
(1) Absorption and emission spectra of the dye overlap so much that self-absorption of the dye arises. The self-absorption of the light emitted from the dye would decrease fluorescence quantum yield of the dye (generally about 0.2) and results in self-quenching.
(2) Because Stokes shift of the dye is small, maximum absorption wavelength and maximum emission wavelength of the dye are so close that scattering light of excitation light would interfere with detection results.
(3) In order to avoid the interference from the scattering light, maximum absorption wavelength cannot be adopted as excitation wavelength or detection vavelength cannot be fixed at maximum emission wavelength, which would decrease sensitivity of detection.
There are two literatures in which it was reported that small Stokes Shift of the dye would affect the detection: (1) Tolosa, L.; Nowaczyk, K.; Lakowicz, J. an Introduction to Laser Spectroscopy, 2nd ed.; Kluwer: New York, 2002. (2) Zhang Z., Achilefu S. Synthesis and Evaluation of Polyhydroxylated Near-Infrared Carbocyanine Molecular Probes. Org. Lett. 2004, 6(12): 2067-2070. To solve the above-mentioned problems, it is very meaningful to develop a fluorescent dye with excellent photophysical properties. It was reported that, when an amino group was introduced to the central position of heptamethine cyanine dye, Stokes shift of the dye could be enlarged from about 20 nm to more than 140 nm, and the fluorescence quantum yield of the dye was also enhanced. However, the longer the conjugated chain of the cyanine dye is, the worse the photostability thereof is, which restricts the further application of heptamethine cyanine dye.
Pentamethine cyanine dye is more stable than heptamethine cyanine dye, but also exists some problems for pentamethine cyanine dye in bio-application. For example:
(1) Small Stokes shift of the dye would also cause above interferences.
(2) Most of polymethine cyanine dyes are structurally symmetrical and lack of single reactive site for fluorescent labeling. In fluorescent labeling, it is better for fluorescent cyanine dye to contain a single reactive group (such as carboxyl group and the like) in the molecule for a special derivation reaction to obtain an ideal fluorescent probe molecule. In order to obtain such polymethine cyanine dye with a single carboxyl group, the usual method is to synthesize an asymmetrical cyanine dye and to intruduce a carboxyl group at one end of the molecule. However, the method makes it troublesome and difficult to synthesize and isolate the dye.
Pentamethine cyanine dye is a near-infrared cyanine dye with the shortest wavelength, and has a much better photostability than heptamethine cyanine dye. Therefore, in the present invention, a series of representative pentamethine cyanine dye with N-substituting at β-position (IIa-g) were firstly synthesized, and then their photophysical properties (spectrum and photostability) were detected, and finally noncovalent interaction of the dyes with protein was investigated.